The present invention relates generally to methods for production of transforming growth factor xcex2 (TGF-xcex2) and, more particularly, to methods for purification of transforming growth factor xcex2 from mammalian bone tissue.
Transforming growth factor xcex2 (TGF-xcex2) proteins are multi-functional cytokines active in regulating growth, differentiation, and morphogenesis in a wide variety of both normal and neoplastic cell types. These growth factors share related biological activities such as chemotactic attraction of cells, promoting cell differentiation and cartilage- and bone-growth induction. TGF-xcex2 is a 25,000 MW homodimer consisting of two 12,500 subunits bound together by nine disulphide bridges. There are three potential N-linked glycosylation sites in the precursor portion of the protein, all of which appear to be used. At least five different forms of TGF-xcex2 (TGF-xcex2 1 to TGF-xcex2 5) have been identified and the sequence homology between the different forms of the mature TGF-xcex2 peptides range from 64-82%. The sequence homology between the precursor sequences is somewhat lower, averaging about 40%. All are functionally homologous although there is some difference in TGF-xcex2 receptor binding properties. TGF-xcex2 has been shown to be an effective cell growth promoter, particularly with epithelial cells, and the use of TGF-xcex2 as a wound healing agent has been demonstrated. It would therefore be desirable to provide methods for producing TGF-xcex2 in relatively large quantities. It would be particularly desirable to provide methods for the purification of TGF-xcex2 from mammalian tissue.
Bones include many proteins, some of which induce or promote bone growth. A great deal of research has been directed to producing specific osteoinductive proteins primarily by recombinant DNA techniques or by purification of the naturally occurring proteins. Such proteins and a variety of processes for obtaining them are the subject of numerous patents. However, very little work has been directed to the economic, large scale commercial production of the naturally occuring protein.
Collagen Corporation of Palo Alto, Calif. is the assignee of a number of patents directed to osteoinductive proteins. U.S. Pat. No. 4,434,094 by Seyedin et al., issued Feb. 28, 1984 identifies a process to partially purify an osteogenic factor and isolate a non-fibrous protein having a molecular weight less than 30 kilodaltons (kD) from demineralized bone extract using cation exchange chromatography. A partially purified bone-inducing factor of 10 to 30 kD and the purification process including extraction from demineralized bone, gel filtration, and cation exchange chromatography on a carboxymethyl cellulose column, and which may include reverse phase-high performance liquid chromatography (HPLC), is described in U.S. Pat. No. 4,627,982 by Seyedin et al., issued Dec. 9, 1986. Also by Seyedin et al., U.S. Pat. No. 4,774,228 issued Sep. 27, 1988, describes two 26 kD proteins found in bone having activity in a TGF-xcex2 assay and purified using a process similar to that taught in Seyedin""s ""094 patent but including reverse phase HPLC or acetic acid-urea gel electrophoresis, where the purified proteins exhibit chondrogenic activity (purportedly related to bone formation). U.S. Pat. No. 4,863,732 by Nathan et al., issued Sep. 5, 1989 is directed to an injectable solution of an osteogenic factor such as that described in Seyedin""s ""982 patent, combined with atelopeptide collagen and further purified by coprecipitation. Other patents relate to mixtures of atelopeptide collagen material, e.g. U.S. Pat. No. 4,789,663 by Wallace et al., issued Dec. 6, 1988 and U.S. Pat. No. 4,795,467 by Piez et al. issued Jan. 3, 1989.
U.S. Pat. No. 4,804,744 by Sen, issued Feb. 14, 1989 identifies a primary osteogenic protein (P3) with a molecular weight of about 22 to 24 kD. This patent also identifies proteins P2 and P4 which are nonosteogenic without P3, and further identifies a method for isolating P3 from demineralized bone tissue including extraction, dialysis, gel filtration and HPLC steps.
U.S. Pat. Nos. 5,290,763, 5371,191 and 5,563,124 to Poser et al., Poser et al., and Damien et al. respectively describe isolation of a growth factor mixture by a multi-step procedure which preferably includes preparing the bone for extraction of proteins by grinding, cleaning, and demineralizing the bone, extracting the bone proteins, and concentrating the extracted proteins by multiple purification steps.
There is a need for a process to efficiently isolate TGF-xcex2 proteins from a natural source on a commercial scale. It would also be beneficial if such proteins could be produced in a manner designed to minimize degradation of such proteins while maximizing production. It would also be beneficial if such proteins could be produced using relatively well-known unit operations in a process which is tolerant of minor variations in process conditions.